Biodegradability of diesel and biodiesel blends

Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2008 Academic Journals

Full Length Research Paper

Biodegradability of diesel and biodiesel blends

Adriano Pinto Mariano*, Richard Clayton Tomasella, Luciano Marcondes de Oliveira, Jonas

Contiero and Dejanira de Franceschi de Angelis

Departamento de Bioquímica e Microbiologia - Instituto de Biociências (IB) - Universidade Estadual Paulista (UNESP).

Accepted 16 February, 2008

The biodegradability of pure diesel and biodiesel and blends with different proportions of biodiesel (2%

(commercial); 5% and 20%) was evaluated employing the respirometric method and the redox indicator

2,6-dichlorophenol indophenol (DCPIP) test. In the former, experiments simulating the contamination of

natural environments (soil from a petrol station or water from a river) were carried out in Bartha

biometer flasks (250 ml), and used to measure the microbial CO

production. With the DCPIP test, the

capability of three inocula to biodegrade the blends was tested. Results show that although biodiesel is

more easily and faster biodegraded than diesel oil, among the blends evaluated (2%, 5% and 20%), only

the blend with higher concentration of biodiesel presented biodegradability significantly different from

diesel and it was not verified an improvement on the biodegradation of the diesel by means of

co-metabolism.

Key words: Biodiesel, diesel, blend, biodegradability, bioremediation.

INTRODUCTION

The introduction of the biodiesel into the Brazilian

ener-getic matrix was determined by a federal law that

establishes a compulsory blend of 2% of biodiesel in

mineral diesel as of 2008 and 5% as of 2013. Currently

Brazil produces approximately 750 million litres of

biodiesel per year, a figure very close to the 840 million

litres necessary to accomplish the 2% blend. Although

either fuel presents the same function, they have very

distinct origins and compositions. Biodiesel is composed

of methyl or ethyl esters of fatty acids with low structural

complexity as oleate, palmitate, estearate, linoleate,

myri-state, laureate and linolenate, derived from different

vegetable oil sources such as soybean, sunflower,

pea-nut, cotton, palm oil, cocopea-nut, babassu and castor oil and

from animal fat (Pinto et al., 2005). Differently, diesel oil

contains 2000 to 4000 hydrocarbons, a complex mixture

of normal, branched and cyclic alkanes, and aromatic

compounds obtained from the middle-distillate fraction

during petroleum separation (Gallego et al., 2001).

Besides the recognised environmental benefits related

to the biodiesel combustion (less emissions of CO

, CO,

*Corresponding author. E-mail: adrianomariano@yahoo.com.br. Fax: +55-1935264176.

SO

, volatile organic compounds and particulate material)

(Pinto et al., 2005), the difference between the fuels

compositions also influences their biodegradability. As

occurs to the diesel oil, the commercialization of biodiesel

or the biodiesel/diesel blend may cause environmental

damages due to spills. The clean-up of these

conta-minated areas can be achieved with bioremediation, a

technique based on the action of microorganisms, which

turn hazardous contaminants into non toxic substances

as CO

, water and biomass. Here again, biodiesel

pre-sents advantages, since studies demonstrate that

biodiesel is more easily biodegraded and less toxic than

diesel oil (Koo-Oshima et al., 1998; Zhang et al., 1998;

Makareviciene and Janulis, 2003; Pasqualino et al.,

2006; Lapinskiené et al., 2006; Khan et al., 2007).

Moreover, some of these works also show that biodiesel

can promote and speed up the biodegradation of diesel

by means of co-metabolism.

Table 1. Soil sample characteristics.

Parameter Value Parameter Value

pH (CaCl2) 6.7 K (mmolc/dm3_{) 1.1}

Organic carbon (%) 0.29 Ca (mmolc/dm3) 15

Total nitrogen (%) 0.02 Mg (mmolc/dm3) 2

Available phosphorus (ppm) 2.0 H+Al (mmolc/dm3) 10

C:N:P 100 : 6.89 : 0.10 Al (mmolc/dm3_{) -}b

Moisture content (%) 12.7 CECa (mmolc/dm3) 28.7

Grain size distribution (%) Sand Silt Clay

81.4 7.3 11.3

Micronutrients(ppm) Heavy metals (ppm)

S Na Fe Mn Cu Zn B Co Mo Ba Cd Cr Ni Pb

12 13 19 3.0 0.6 7.3 0.15 0.56 -b 4.06 0.12 9.93 0.30 7.10

a_{Cation exchange capacity} b_{Not detected}

Table 2. Water sample characteristics.

Parameter Value Parameter Value Parameter Value

pH 7.38 Ammonia (mg/L) 1.65 Bacteria (CFU/mL) 2.6 . 103

BOD (mg/L) 3.33 Chlorate (mg/L) 5.9 Filamentous Fungi (CFU/mL)

17 COD (mg/L) 29.12 Cyanate (mg/L) 0.006

DO (mg/L) 6.58 Phenols (mg/L) - a Yeast (CFU/mL) 3

Conductivity ( S/cm) 82.9 Volatile solids (mg/L) 0.045 Toxicity (EC50)b _- a Acidity (mg/L) 7.76 Fixed solids (mg/L) 0.092

Alkal. HCO3 (mg/L) 23.21 Soluble solids and in

0.137 Nitrite (mg/L) 0.043 suspension (mg/L)

Nitrate (mg/L) 1.009 Sedimentation <0.1

a_{Not detected} b

Daphnia similis

MATERIAL AND METHODS

Soil and water sampling and their characteristics

The soil sample was collected at a petrol station (Rio Claro/SP/Brazil) during the replacement of underground pipes (0.50 m depth). This sample showed low level of contamination (104 mg/Kg) by unknown fuel, possibly due to leaks in the pipes and ground infiltrations (Mariano et al., 2008a,c). Until performing the biodegradation experiments, the sample was stored at 5o_{C. Table 1}

summarizes some of the soil physicochemical characteristics. Values of heavy metals concentrations are not above the more res-tricted levels set by the Cetesb (São Paulo Environmental Agency – Brazil) and by the Dutch list (Cetesb, 2005).

The soil physicochemical analyses were performed by the labo-ratory “Instituto Campineiro de Análise de Solo e Adubo (ICASA)”, according to the methodology proposed by Embrapa (1997), except the following parameters; total nitrogen (laboratory “PIRASOLO – Laboratório Agrotécnico Piracicaba”, according to Embrapa (1997)) and the moisture content (obtained by the oven drying method).

The water sample was collected at the Jaguari river (22o _{42' 00"}

S / 47o _{08' 06" W) located in Paulínia (SP/Brazil). The composite}

sample was obtained at the river surface along a transect perpen-dicular to the flow direction. Nearby the sampling location, an oil

refinery (Replan/Petrobras) and roads represent potential sources of contamination. Table 2 summarizes some of the water characteristics (APHA, 1998).

Respirometric experiment

Biodegradation experiments simulated soil and water contamina-tions, respectively from the petrol station and the Jaguari river. The soil and water contamination was carried out by adding fuel (50 ml/Kg of soil; 10 ml/L of water) with the following volume percent compositions of the biodiesel/diesel blend: 0/100; 2/98; 5/95; 20/80 and 100/0, respectively denominated: B0, B2, B5, B20 and B100. The diesel oil and the B2 blend were obtained at petrol stations (respectively, BR and ALE distributors) in Rio Claro (SP/Brazil). The other blends were prepared in laboratory combining the diesel oil with a biodiesel produced from castor oil.

Biodegradation experiments were carried out in Bartha biometer flasks (250 ml) used to measure the microbial CO2 production

(Bartha and Pramer, 1965; Régis and Bidoia, 2005; Mariano et al., 2007a, 2008a,b,c). The CO2 produced is proportional to the

car-bons consumed by microorganisms from the test substrate. Thus, the amount of CO2 is proportional to the percentage of substrate

0 10 20 30 40 50 60 70 80

0 20 40 60 80 100 120

time (days)

C

O

2

d

ia

ry

p

ro

d

u

ct

io

n

(µ

m

o

l.d

-1 ) control

B0 B5 B20 B100

Figure 1. Daily CO2 production during incubation of the first

respirometric experiment with the soil contamination.

0 1000 2000 3000 4000 5000 6000 7000

0 20 40 60 80 100 120

time (days)

cu

m

ul

at

iv

e

C

O

2

p

ro

du

ct

io

n

(µ

m

ol

) _control

B0 B5 B20 B100

Figure 2. Cumulative total amounts of CO2 produced by the first

respirometric experiment with the soil contamination during incubation. Each error bar represents 1 SD of three replicate.

total CO2 production can provide excellent information on the

biodegra-dability potential of hydrocarbons (Balba et al., 1998). CO2

evolution measures ultimate degradation (mineralization) in which a substance is broken down to the final products, while, for instance, the gas chromatography (GC) analysis measures primary degrada-tion only in which the substance is not necessarily transformed to the end products.

For each experimental condition, the biometer flasks were pre-pared in triplicates (3 x 50 g of soil or 50 ml of water) and incubated at 27o_{C in the dark. The CO}

2 produced was trapped in a 10.0 ml

solution of KOH (0.2 N), located in the side-arm of the biometer. This solution was periodically withdrawn by syringe, and the amount of CO2 absorbed was then measured by titrating the

residual KOH (after the addition of barium chloride solution (1 ml; 1.0 N) used to precipitate the carbonate ions) with a standard solution of HCl (0.1 N). During this procedure, the biometers were aerated during 1.5 min through the ascarite filters.

Biodegradability test – DCPIP indicator

The biodegradability of the biodiesel/diesel blends was also verified using the technique based on the redox indicator 2,6-dichlorophe-

0 20 40 60 80 100

0 20 40 60 80 100

time (days)

C

O

2

d

ia

ry

p

ro

du

ct

io

n

(µ

m

o

l.d

-1 ) control

B0

B2 (commercial)

Figure 3. DailyCO2 production during incubation of the second

respirometric experiment with the soil contamination.

nol indophenol (DCPIP) (Hanson et al., 1993). The principle of this technique is that during the microbial oxidation of hydrocarbons, electrons are transferred to electron acceptors such as oxygen, nitrates and sulphate. By incorporating an electron acceptor such as DCPIP to the culture medium, it is possible to ascertain the ability of the microorganism to utilize hydrocarbon substrate by observing the colour change of DCPIP from blue (oxidized) to colourless (reduced). This Hanson et al. (1993) technique has been employed in several works (Cormack and Fraile, 1997; Roy et al., 2002; Mariano et al., 2007b, 2008a,c,d), but for the first time, this technique was used to evaluate the biodegradability of biodiesel/diesel blends.

The capability of three inocula to biodegrade the blends B0, B2, B5, B20, B50 and B100 was tested: consortium 1 (obtained from the soil at the petrol station); consortium 2 (from an uncontaminated soil collected at UNESP campus) and the culture Pseudomonas

aeruginosa LBI (Benincasa et al., 2002).

The inoculum P. aeruginosa LBI was prepared using bacterial

cells transferred from the storage culture tubes and streaked onto the surface of Petri dishes containing PCA medium (Acumedia, EUA). To prepare the other two consortia, 10 g of respective soils were added to Erlenmeyer flasks (125 ml) containing 50 ml of sterile saline solution and kept under agitation for 1 min. After this period, the saline was streaked onto the surface of Petri dishes containing PCA medium. The Petri dishes were incubated during 24h at 35o_{C and then cells were harvested using sterile saline}

solution.

The inocula (1.0 ml, concentration not determined) were added to Erlenmeyer flasks (250 ml, duplicates) that contained sterile Bushnell-Hass (BH) medium (50 ml) and 1% (v/v) of the blends. The concentration of DCPIP was 20 mg/ml. Erlenmeyer flasks were kept under agitation (84 rpm) at 35.0 ± 0.5o_{C. The BH medium}

consists of, gL-1_{: MgSO}

4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; K2HPO4,

1.0; NH4NO3, 1.0; FeCl3, 0.05 (Difco, 1984).

RESULTS

0 1000 2000 3000 4000 5000

0 20 40 60 80 100

time (days)

cu

m

ul

at

iv

e

C

O

2

p

ro

d

uc

tio

n

(µ

m

ol

)

control B0

B2 (commercial)

Figure 4. Cumulative total amounts of CO2 produced by the second

respirometric experiment with the soil contamination during incubation. Each error bar represents 1 SD of three replicate.

0 10 20 30 40 50

0 10 20 30 40 50

time (days)

C

O

2

d

ia

ry

p

ro

d

u

ct

io

n

(µ

m

ol

.d

-1 ) controle

B0

B2 - commercial B5

B20 B100

Figure 5. Daily CO2 production during incubation of the

respirometric experiment with the water contamination.

and 2) the total CO

produced (µmol/ (Kg/day)) from B0,

B5, B20 and B100 were 804.4, 882.6, 911.7 and 1114.9,

respectively. In the second experiment (Figures 3 and 4),

these values for B0 and the blend B2 (commercial) were

1034.5 and 1069.3, respectively. These values and the

curves in the graphics show that the CO

production

increased as more biodiesel were present in the blend.

Statistically (Anova, p=0.05) only the blend B20 and the

pure biodiesel (B100) differed from B0.

The daily and cumulative CO

productions during the

respirometric experiment with the water contamination

are shown in Figures 5 and 6, respectively. The total CO

produced (µmol/(Kg/day)) from B0, B2, B5, B20 and

B100 were 287.1,; 287.5, 334.0, 367.8 and 466.4,

res-pectively. Again, as in the soil contamination, these

values and the curves in the graphics show that the CO

production increased as more biodiesel was present in

the blend. Statistically (Anova, p=0.05) only the blend

B20 and the pure biodiesel (B100) differed from B0.

0 200 400 600 800 1000 1200

0 10 20 30 40 50

time (days)

cu

m

ul

at

iv

e

C

O

2

p

ro

du

ct

io

n

(µ

m

o

l) _control

B0

B2 - commercial B5

B20 B100

Figure 6. Cumulative total amounts of CO2 produced by the

respirometric experiment with the water contamination during incubation. Each error bar represents 1 SD of three replicate.

The results obtained with the biodegradability test using

the redox indicator DCPIP (Table 3) show that the time

necessary to decolourization of the DCPIP indicator

decreased with the increase of the concentration of

biodiesel in the blend.

DISCUSSION

Previous works related to the biodegradation of biodiesel

and diesel blends mainly focused on the water

conta-mination (Zhang et al., 1998; Makareviciene and Janulis,

2003; Pasqualino et al., 2006) with the exception of the

work by Lapinskiené et al. (2006), which evaluated the

microbial transformation of these compounds in soil.

These works demonstrated that biodiesel and the

biodiesel/diesel blends are more easily and faster

bio-degraded than diesel oil. In the present work, similar

results were obtained now with the contamination of soil

from a petrol station and water from a river. Experimental

evidences are the CO

production in the respirometric

experiments that increased as more biodiesel was

present in the blend and the time necessary for

de-colourization of the indicator in the biodegradability test

using the redox indicator DCPIP, which decreased with

the increase of the concentration of biodiesel in the

blend.

Table 3. Time (in hours) to decolourization of the DCPIP indicator.

Microorganism

first experiment second experiment

B0 B5 B20 B50 B100 B0 B2 (commercial)

consortium 1a 36 29 27 24 22 120 90

consortium 2b _{68 44 24 20 20 64 41}

P. aeruginosa LBI 28 13 9 8 3 72 20

a_{From the soil at the petrol station} b_{From a non-contaminated area}

Obs: During the test, no decolourization of the substrate control (without inoculum) or of the inoculum control (without oil) was observed.

0 1000 2000 3000 4000 5000 6000 7000

0 20 40 60 80 100 120

time (days)

cu

m

ul

at

iv

e

C

O

2

p

ro

du

ct

io

n

(µ

m

ol

)

B0

B20 (experimental data) B20 (linear combination) B100

Figure 7. Evaluation of the synergic effect (co-metabolism) for the blend B20 (first respirometric experiment with the soil contamination).

Moreover, the presence of aliphatic cyclic hydrocarbons,

polycyclic aromatic hydrocarbons (PAHs) and

alkylben-zenes, as well as their derivatives such as toluene,

xylenes and PCBs (phenyl and biphenyls) gives the

diesel a composition much more chemically complex.

Another point to be discussed is that Zhang et al.

(1998) and Pasqualino et al. (2006) verified that biodiesel

can promote and speed up the biodegradation of diesel

by means of co-metabolism, that is a term used to

des-cribe the process in which microorganisms use a second

substrate (readily degradable) as the carbon (energy)

source to degrade the first substrate which otherwise is

scarcely attacked by the microorganisms when it is the

sole carbon source. Based on this concept, researchers

verified that in some cases biodiesel can be applied in

contaminated areas as an enhancement agent to

bio-remediation processes (Mudge and Pereira, 1999; Taylor

and Jones, 2001; Obbard et al., 2004 and

Fernández-Álvarez et al., 2006, 2007).

To determine how biodiesel can improve the

bio-degradability of the pure diesel, the synergic effect was

evaluated for the mixtures according to the methodology

proposed by Pasqualino et al. (2006), that is based on

the measurement of CO

in a respirometric experiment.

The total amount of CO

(cumulative value) produced

with a certain blend (B2, B5 and B20) was compared to a

linear combination (LC) (Equation 1) of the total amount

of CO

produced with the pure compounds (B0 and

B100), as follows:

LC = D.(CO

)

+ B.(CO

)

(1)

Where D is the percentage of diesel in the blend, B the

percentage of biodiesel in the blend, (CO

)

the total

amount of CO

(cumulative value) produced with B0 and

(CO

)

the total amount of CO

(cumulative value)

produced with B100.

This linear combination was compared with the

experimental values of the blends during the days of the

experiments. Figure 7 shows the results for the soil

con-tamination with the blend B20. This case exemplifies

what was observed for all the other blends considering

both soil and water contaminations. According to the

methodology of analysis adopted, the curves of the

experimental data and that of the linear combination are

coincident, it indicates that, the biodiesel did not improve

the biodegradation of the diesel by means of

co-metabo-lism. It is important to comment that this methodology of

analysis has limitations since it is not based on

chromatographic analysis, which could indicate that only

the biodegradation of certain compounds present in the

diesel could be favoured by the co-metabolism as

observed by Fernández-Álvarez et al. (2007).

The results obtained with the biodegradability test using

the redox indicator DCPIP also indicate that the soil of

the petrol station (from where was obtained consortium 1)

had a microbiota adapted to degrade the fuels and the

tests with consortium 2 show that the presence of

hydrocarbonoclastic microorganisms in soils is

ubiqui-tous, even in unpolluted soils (Venkateswaran and

Harayama, 1995; Ron and Rosenberg, 2002 apud Lee et

al., 2006; Mariano et al., 2008c).

The time demanded by inoculum 1 for the

decolouri-zation of the blend B0 in comparison to B5 decreased

19.4% and for

P. aeruginosa

LBI, 53.6% (Table 3). This

mixed cultures, commensalisms play an important role

since each species may have a specific function in the

enzymatic reaction sequences, responsible for the

breakdown of more complex molecules. Thus, consortia

have less difficulty in biodegrading diesel. This fact is in

agreement with the respirometric data, where the CO

produced with the blends B2 and B5 did not differ

significantly from the pure diesel (B0).

Conclusion

Although biodiesel is more easily and faster biodegraded

than diesel oil, among the blends evaluated (2%, 5% and

20%), only the blend with higher concentration of

bio-diesel presented biodegradability significantly different

from diesel and it was not verified an improvement on the

biodegradation of the diesel by means of co-metabolism.

In natural environments, as considered in this work, the

commensalisms between different communities of

microorganisms facilitates the biodegradation of diesel

oil, for this reason, the addition of low quantities of

biodiesel (2% or 5%) may not represent a gain when the

biodegradability aspect is concerned.

AKNOWLEDGMENT

The authors acknowledge the

Agência Nacional do

Petróleo, Gás Natural e Biocombustíveis

(ANP)

(PRH-05).

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